Abstract: Pre-messenger RNA (pre-mRNA) splicing is a process in which introns are removed to join exons together. This process is used during the synthesis of nearly all metazoan mRNAs (~93% of human mRNAs) and is an important means of regulating gene expression. With only a handful of exceptions, splicing occurs in cis - the exons that are joined together are located on the same pre-mRNA. In some eukaryotes, including nematodes, trypanosomes and planarians, splicing can occur in trans. In these cases, a specialized spliced leader RNA is spliced to the 5' end of protein coding RNAs. Interestingly, there are a few cases where a distinct type of trans-splicing has been shown to occur, namely, the splicing of exons from protein coding genes. The two best characterized examples are the mod(mdg4) and lola genes from Drosophila. In each of these genes, a group of common 5' exons can be spliced in trans to one of several (26 for mod(mdg4) or 22 for lola) variable 3' exons. One convincing case of trans-splicing has been shown to occur in mosquitos and one has been recently shown in humans. In each of these cases, trans-splicing of these genes was discovered fortuitously. As a result, the true extent of trans-splicing is unknown. Moreover, nothing is known about the mechanisms involved in trans-splicing. We have recently used deep sequencing to identify 80 new genes that are trans-spliced in Drosophila. The long- term goals of this project are to further explore the trans-splicing landscape in Drosophila and to determine the mechanisms involved in this process. We will first perform an exhaustive survey for trans-spliced genes throughout development in different Drosophila species. Second, we will perform experiments to test whether the trans-spliced mRNAs are translated and are functional. Finally, these experiments will be complemented by a combination of biochemical, genetic, genomic, cell biology, and bioinformatics experiments designed to determine the mechanisms by which trans- splicing occurs. Together these experiments will provide tremendous insight into the mechanisms of trans-splicing, a completely understudied yet important process. Given the potential utility of trans-splicing in treating human diseases and that trans-spliced mRNAs in humans have recently been linked to cancer, it is likely the discoveries we make will be of direct relevance to human health.